During the current funding period, Dr. Wucherpfennig's lab has developed novel approaches for the quantification of autoantibodies and CD4 T cells in autoimmune diseases. This work was conducted as integral component of research projects, and the technologies have now matured to a point where these reagents can be generated most efficiently by a core supervised by Dr. Wucherpfennig. Collaborative work between the Wucherpfennig and Hafler labs resulted in the creation of autoantigen tetramers that enable sensitive detection of autoantibodies in human demyelinating diseases. Tetramers of myelin oligodendrocyte glycoprotein (MOG) were shown to permit sensitive detection of MOG autoantibodies in patients with acute demyelinating encephalomyelitis (ADEM) and pediatric MS. The first major aim for this core will be to generate such antigen tetramers for quantification of autoantibodies against other selfantigens in MS and type 1 diabetes. The core will generate both radiolabeled versions for autoantibody quantification as well as fluorescent versions for characterization and isolation of autoantigen-specific B cells in patients with MS and type 1 diabetes, and in relevant animal models. During the present funding period, the Wucherpfennig lab has also developed a novel approach for the creation of MHC class II tetramers. MHC class II molecules are expressed with a covalently bound CLIP peptide that protects the binding site during biosynthesis and purification. The low affinity CLIP peptide is rapidly released following linker cleavage, and these MHC class II/CLIP precursors can therefore be used for the generation of a variety of different MHC class II tetramers from a single protein preparation. This approach enabled quantification of autoantigen-specific CD4 T cells in animal models of MS and type 1 diabetes. In particular, it allowed simultaneous visualization of regulatory and effector T cells in Foxp3-GFP knock-in mice during the course of experimental autoimmune encephalomyelitis (EAE). The second aim for this core will therefore be to generate MHC class II tetramers for all projects of this PPG and to further develop this technology by generation of multimers with higher valency that can be used to detect lower affinity CD4 T cell populations.